26 X)SL/ SLiRI•I 1 I I , YRL P I I

4.2. Rapid preparation of plasmid by restriction endonuclease cleavage, elec-DNA from the positive clones. trophoretic analysis in agarose gels and

4.3. Restriction endonuclease diges- "Southern blotting" techniques ("Charac-tions. terization of albumin cDNA containing plas-

4.4. Agarose gel electrophoresis. mids": Part III).4.5. Transfer of DNA from gels to ni-

trocellulose filters ("Southernblots"). 2. Construction of a cDNA library

4.6. Blot hybridization to rat albumin of messenger RNA species from rat liver32P-cDNA.

5. Addendum5.1. Buffers.5.2. Culture media.5.3. Restriction map of pBR322.5.4. Safety considerations: Physical

containment.

6. Bibliography

2.1. Introduction

The DNA copy of an mRNA moleculesynthesized by the avian myeloblastosis virus(AMV) reverse transcriptase is termed "com-plementary DNA" (generally abbreviated to"cDNA"). The term cDNA clone is nowused to describe a bacterial cell transformedby a plasmid containing the DNA copy of anRNA molecule. A "cDNA clone bank" or"cDNA library" from a given cell or tissue isa population of bacterial transformants,each containing a plasmid with a singlecDNA insert, complementary to one of thecells mRNA molecules. A complete cDNAlibrary will contain a sufficiently large num-ber of individual transformants such thatevery mRNA species is represented at leastonce in the bacterial population.The preparation of a cDNA library can be

carried out by various procedures which dif-fer in the enzymatic steps used to prepare re-combinant plasmid DNA for the transfor-mation of competent E. coli cells. The proce-dure we will use in this course is summarizedin Figure 1. The first step is the synthesis ofDNA copies of the mRNA molecules presentin the polyadenylated RNA preparation,using reverse transcriptase. Following alkali-ne hydrolysis of the RNA, the single-stranded cDNA will serve both as templateand primer for the synthesis of the secondstrand which is covalently linked to the first.The loop of the hairpin molecule formed canbe specifically cleaved with single-strand-specific S, nuclease. The double-strandedcDNA (ds-cDNA) will be inserted into the

1. Objective

The aim of this course is to introduce youto current techniques used to construct re-combinant DNA clones containing specificgenes and to screen a large number of E. colicolonies for recombinant plasmids carrying aspecified DNA sequence.Two approaches can be used to construct

recombinant DNA clones containing specificgene sequences; these are direct cloning ofgenomic DNA fragments or cloning of adouble-stranded cDNA made from themRNA. In this course we will construct acollection of recombinant DNA moleculeswhich will permit the cloning of a largesample of DNA sequences complementary torat liver mRNA species ("Construction of acDNA library": Part I).The cDNA library will be screened for in-

serts complementary to rat albumin mRNAby in situ colony hybridization using a `P-labelled rat albumin cDNA probe ("Screen-ing for rat albumin cDNA sequences":Part II).The recombinant plasmids bearing albu-

min cDNA sequences will be characterized

bacterial plasmid pBR322. This vector

carries genes coding for resistance to ampi-

cillin and to tetracycline. Insertion of a fo-

reign DNA molecule into one of these genes

will destroy the resistance capacity to the an-

tibiotic. This offers a convenient first hand

screening procedure for identifying recombi-

nant clones.

To construct hybrid molecules, the

pBR322 is linearized by the restriction endo-

nuclease Psti which cleaves the DNA only

once, and is tailed with oiigo(dG) by the

terminal deoxynucleotidyl transferase. The

ds-cUNA is similarly tailed with oligo(dC)

and annealed to the plasmid DNA. Follow-

ing transformation in E. coli competent cells

(MC10o1), the gaps are completed in the

bacterial cell and in this process, the PstI si-

tes are reconstructed. The resistance to ampi-

cillin is however lost. This cloning procedure

which allow excision of the inserted

cDNA sequence by restricting the recombi-

nant clone with PstI, is described in detail

below.

2.2. Synthesis of complementary DNA

to total polyadenylated polysomal RNAfrom rat liver

^^KOroco^s o^tnccti^f^E^ki..a cr,v^^nc:a 27

43°C for 60 min the reaction is terminated by

the addition of 10 µl of SDS 10 % and 10 µl

of EDTA 0.5 mM. The template RNA is

then hydrolyzed making the mixture 0.3 M in

NaOH and incubating at 70°C for 60 min.

The solution is neutralized, an aliquot is re-

moved for TCA precipitation and unincor-

porated nucleotides are removed by Sepha-

dex G-100 chromatography. The excluded

fractions are monitored by Cerenkov count-

ing and are pooled. The cDNA is then preci-

pitated with 2 volumes of ethanol at -20°C

overnight.

2.3. Double-stranded cDNA synthesis

The precipitated single-stranded cDNA is

collected by centrifugation (10,000 xg for

90 min), washed with ethanol, dried and dis-

solved in 50µl of EDTA 0.5 mM. An aliquot

(1 µl) is removed for scintillation

counting.

Second-strand synthesis is carried out in

sterile tubes in a final volume of 125 µl with

reverse transcriptase. Reaction conditions

are the same as those used for the first-strand

except that this time oligo (dT) and actino-

mycin Dare omitted and 'H-labelled dCTP(100 µM; 5 Ci/mmole) is used. The reactionis stopped as indicated above with EDTA

(10 µl EDTA 0.5 M) and SDS (5 µl SDS

10 %). An aliquot is removed for TCA pre-

cipitation and unincorporated nucleotides

are removed by Sephadex G-100 chromato-

graphy. The excluded fractions are monito-

red by scintillation counting and are pooled.The ds-cDNA is then precipitated with 2 vo-lumes of ethanol at -20°C overnight.

2.4. Cleavage of the hairpin loopand removal of single-stranded.sequences

The ethanol precipitate is collected bycentrifugation (10,000 xg for 90 min),

28 JOSEP MARIA SALA i TRPPAI

washed twice with ethanol, dried and dis-

solved in 202 Ml of EDTA 0.5 mM. A 2 MI ali-

quot is removed for scintillation counting

and to the remainder the following are ad-

ded: 2.5 Ml of 4 M Na Acetate buffer pH 4.6:

34 Ml of 2 M NaCl; and 12.5 Ml of 100 mM

ZnSO4. The mixture is then incubated with

1500 units of S, nuclease (Miles Research

Products Ltd.) at 25°C for 60 min. The reac-

tion is stopped with 25 MI of 1 M Tris-HCI,

pH 8.4, and an aliquot is TCA precipitated.

The remainder is extracted with one volume

of phenol: chloroform (1:1) and the aqueous

phase is precipitated with 2 volumes of

ethanol.

2.5. Addition of homopolymer dC tracts

to the 3'-ends of double -stranded

cDNA

The terminal addition of dCTP to the ds-

cDNA by terminal deoxynucleotidyl + trans-

ferase is carried out in the presence of Coe'instead of Mgt ions, which allows the enzy-

me to accept double-stranded DNA as a pri-

mer instead of single-stranded DNA, as un-

der the usual assay conditions (Roychou-

dhury et al., Nucleic Acid Res. 3:101-116,

1976).The double-stranded cDNA is collected by

centrifugation, washed twice with ethanoland dissolved in a small volume of EDTA0.1 mM. The reaction is carried out in anEppendorf tube and contains in a final volu-me of 25 l:- 140 mM potassium cacodylate.- 30 mM Tris base pH 6.9.- 0.1 mM dithiothreitol.- I mm Cock.- 0.1 mM (12P)-dCTP 5 Ci/mmole.- 100 Mg/ml double-stranded cDNA.- 25 units of terminal transferase (PL

Biochemicals).The reaction mixture is heated at 37°C

prior to the addition of the enzyme. Add

CoCl2 last, just before the enzyme. The reac-tion is followed by incorporation of labeled

dCTP into TCA-insoluble material. After 5min incubation chill the entire reaction mix-

ture on ice water. Remove 1µl aliquot, TCA

precipitate, count and calculate the number

of bases added per end knowing the molarity

in ends. Aim to add 15-20 nucleotides per 3'-end of DNA. If necessary the reaction can berestarted at 37°C without the addition ofnew enzyme to increase the length of the

tails.

When the required number of nucleotides

has been obtained the reaction is stopped by

the addition of EDTA to 10 mM, followed

by extraction with an equal volume of phe-

nol: chloroform mixture. The aqueous phase

is then passed over a smalll Sephadex G-50

column to separate unincorporated reaction

components from tailed ds-cDNA, using

10 mM Tris-HCI pH 7.4, 0.1 M NaCl and

0.2 mM EDTA as the running buffer. The

void volume is collected and stored at 4.°C.

2.6. Preparation of pBR322 plasmid vector:

Restriction cleavage by Pst/

and terminal addition of dG TP

In order to construct a hybrid plasmid,

pBR322 is first linearized by restriction clea-vage using the endonuclease Pstl and then

tailed with dGTP.Mix in a plastic Eppendorf tube:

- 10 Ml of 10 x restriction buffer (60 mM

Tris-HCI, pH 7.4; 50 mM NaCl, 60 mM

MgC12; 60 mM 2-mercaptoethanol).

- 10 Mg of pBR322 DNA.

- 50 units of Pstl.

- Distilled water up to a final volume of

100 Ml.

Incubate at 37°C for 1 hr. Add another

50 units of restriction endonuclease Pstl and

continue the incubation for a second hour.

The DNA is then extracted with phenol-chlo-

roform and reprecipitated with NaCl and

ethanol.

PROTOCOLS D'ENGI,NY"L'RlA GENET/CA

s ............... ................. LAI^ 3 ARN-cs%age,

t Reverse iranscriptase

............................ [AYE ^ Heteroduplexe

(T7^ s.

1 NaOH

( T^n s• cADN ss

t ADN polymerise I

a

f Nuclease 5^

^'

s'

s' cADNds

Y

(T^n ,

± Pst^

PBR 322

^ ACGTC G s'

s'G CTGCA^'

dCTP dGTP

t Terminole tronsferase ^ Terminole+rrmsfurase

7l)

(C ^_r (G7nACGTC . G

(C^ G .- CTGCACG;^

HY lotion1

-G (Ch--- -^^ACGTG -.

^ _CTGCA(G^1-----1CYrs G __

-- ----

Transformation

G+ACGITC^.T -(G}^ACGTC

CTGCA(L,,, r C]^ T CiCA^^

' '

30 3OSLP,Vaki.-a s.-1L:1 i TRLLPAI

Five µg of Pstl restricted pBR322 are thentailed with dGTP residues in a 50µl reactionvolume composed of 140 mM potassium ca-codylate, 30 mM Tris base (pH 6.9), 0.1 mMdithiothreitol, 1 mM CoClzi 0.2 mM 3H-dGTP (5-10 Ci/mmole), and 50 units of ter-minal transferase. The reaction is carried outat 37°C for the terminal addition of dCTP tothe 3'-ends of double-stranded cDNA. 10-15nucleotides should be added per 3'-end ofpBR322 DNA.

2.7. Annealing to form hybrid plasmidDNA

The molecular weight of pBR322 is about2.6 x 106. If we estimate the average molecu-lar weight of the ds-cDNA preparation to beabout 4.4 x 105, we will need a mass ratio ofapproximately 1:6 for equimolar amounts.17 ng of double-stranded cDNA tailed withpoly(dC) will be annealed with 100 ng ofpBR322 tailed with poly(dG) in 25 µl of10 mM Tris-HCl pH 7.4, 0.1 M NaCl and0.2 mM EDTA. The mixture is treated to63°C for 3 min, transferred to a 43°C waterbath for 2-3 hr, then cooled slowly to roomtemperature.

2.8. Preparation of competent E. coli ells

- Streak out E. coli HB 101 (thr-,1eu-, pro-, recA-, hsdR-, hsdM-) or MC1061 (galU-, galK--, recA-, hsr-, hsm+) onLB agar plate on day preceeding experiment.- Inoculate a single colony into 50 ml of

L broth and incubate, with shaking, at 37°Cuntil the A660 reaches 0.5-0.6 (about 5 x 10'cells /ml).

-- Centrifuge 2 x 10" cells (4 ml) in a 15 mlCorex tube at 8000 g for 10 min at 4°C. Pooroff supernatant and resuspend pellet in 2 mlof 10 mM MOPS pH 7.0, 10 mM RbCl.Cells should be resuspended as gently as pos-sible.

- Centrifuge immediately at 8000 gfor 10 min at 4°C. Resuspend the pelletin 2 ml of ice-cold 100 mM MOPS, pH 6.5,50 mMCaCl,i 10 mM RbCI. Hold cells on icefor 30 min. Treat cells as gently as possibleafter this step.- Centrifuge for 10 min at 8000 g. Drain

tubes throughly on absorbent paper (Kimwi-pes). Resuspend the pellet gently in 0.4 ml ofthe above indicated 100 mM MOPS buffersolution. The competent cells are then usedfor transformation.

2.9. Transformation of E. coli competent

cells

Transformation is carried out by mixing0.2 ml of competent cells with 3 yl of DMSOand 20 yl of the annealed mixture (about100 ng of plasmid DNA). The mixture ishold for 30 min on ice without shaking thenincubated at 43.5°C for 30 sec (heat shock).After dilution at room temperature with2 ml of Z broth, tubes are hold for 60 min at37°C without shaking. 100 pl of the suspen-sion of transformed cells is spread on 9 cm

Petri dishes containing LB medium supple-mented with tetracycline. A control experi-

ment is performed using 100 ng of unan-nealed dG-tailed plasmid vector as donorDNA to determine the degree of backgroundclones arising from intact pBR322 moleculesstill remaining in the preparation.To assess the efficiency of transformation

another control experimental is carried outusing 100 ng of intact pBR322 as donorDNA. In this case a series of dilutions (from10' to 10-6) are to be made from the 2 ml Zbroth before plating. Then 100 yl of the10-4, 10-5 and 10-6 suspensions of transfor-med cells are spread on Petri dishes con-taining LB medium. 100 pl aliquots of the10-1, 10-2 and 10-' suspensions are equallyspread on LB plates supplemented with

tetracycline or ampicillin.

rkoroco^_s c^^i_ ci,^rtHi.^ c,E.tiE^rica 31

2.10. Selection of antibiotic resistant clones

pBR322 carriers markers for ampicillin

and tetracycline resistance. Insertion of fo-

reign DNA into the PstI site of this plasmid

causes inactivation of the beta-lactamase ge-

ne and allows identification of hybrid plas-

mids carrying foreign DNA.

Transformed cells which have taken up

either pBR322 or hybrid plasmid DNA will

be Tc' and will grow on tetracycline con-

taining plates whereas non transformed

E. coli cells will not. From these clones, Ap'

cells (containing recombinant plasmids) are

selected by replica plating. Individual clones

ark transferred using the tip of a sterile

toothpick first to a ampicillin containing pla-

te and then to a tetracycline containing one.

A template is used to align the position of the

transformed cells on the two plates. The pla-

tes are incubated for 18 hours at 37°C. Then

by comparing the two plates colonies which

grow on the Tc containing place but not Ap

containing plates are identified.

in rat liver, albumin cDNA sequences should

be present in a high proportion of the clones

(around 5 %).

We will screen a moderate number of

cDNA clones from the bank with an in vitro

labeled probe derived from purified rat albu-

min mRNA. To illustrate the specificity of

the in situ colony hybridization procedure of

Grunstein and Hogness (Proc. Natl. Acad.

Sci. 72:3961-3965, 1975) we shall prepare

two different radioactive DNA probes: a) A

32P-labeled cDNA made to purified rat albu-

min mKNA by using the AMV reverse trans-

criptase, and b) a'^P-labeled probe made by

nick-translation of pBR322 DNA. The

cDNA wil allow us to select for clones con-

taining an inserted albumin cDNA sequence,

whereas the nick-translated pBR322 DNA

should hybridize to all transformed E. co/i

colonies.

3.2. Preparation of radioactive DNA probes

a. Albumin cDNA synthesis

3. Screening for clones containing specific

DNA sequences : application to ratalbumin cDNA sequences

3.1. Introduction

The preparation of a cDNA bank allows

for the obtention of any particular cDNA

clone for which we have a method of selec-

tion. The term "screening" is normally

applied [o describe any procedure designed

to identify and isolate a particular clone

from the bank.Eucaryotic mRNA sequences are present

at widely varying abundances in different

cell types, and in general, the frequency of

occurrence of a particular clone in a bank is

proportional to its abundance. We have pre-pared a cDNA bank from rat liver polyade-nylated polysomal RNA. Since albuminmRNA is the most abundant mRNA species

32 JOSLP MARDI .SALA i TRLL?/l /

ture 0.3 076 in NaOH and incubating at 70°Cfor 60 min. The solution is neutralized, analiquot is removed for TCA precipitationand unincorporated nucleotides are removedby Sephadex G-50 chromatography. Theexcluded fractions are monitored by Ceren-kov counting and are pooled. The cDNA is

then precipitated with 2 volumes of absolute

ethanol at -20°C overnight.

b. Labeling of pBR322 by nick- translation

The labeling of DNA by nick-translation isbased upon the observation that E. coli DNApolymerase I binds at a nick and, in the pre-sence of the required deoxynucleosidetriphosphates, extends the primer terminus.Nicks in DNA are introduced by limitedDNase I action. Addition of DNA polymera-

se I catalyses then the nick-translation reac-tion by the simultaneous operation of thepolymerase activity (which adds nucleotideresidues at the 3'-hydroxyl terminus) and the

5'-3' exonuclease activity (which removes

5'-phosphate residues from the 5'-terminus).The reaction is carried out in an stelire Ep-

pendorf tube containing:

- 50 mM Tris-HCI pH 7.8.

- 5 mM MgCl,.

- 5 mM DTT.- 10 µM of each dTTP, dGTP.

- 5 yM of each 32P-dATP, 32P-dCTP(500 Ci/mmole).

- 50 µg/ml bovine serum albumine.

- 1 pg of DNA.After addition of 3 yl of I ng/ml of

DNase I (freshly diluted from a stock

I mg/ml DNase solution) the mixture is in-

cubated at 37°C for 10 min and immediately

cooled in ice. 3 µl of E. co/i polymerase I

(5 units/ml) are then added and the mixture

is incubated at 15°C for 2 h. The reaction is

terminated by the addition of 20 yl of 50 mMTris-HCI pH 8.0 containing 25 mM EDTA

and 0.5 % SDS. The solution is then made

0.3 M in NaOH and boiled for 2 min, fo-

Ilowed by immediate cooling in ice. Micro-coccal DNA carrier (20 µg) is added and themixture is neutralized with I M acetic acid.

A small aliquot (I µl) is removed for TCA

precipitation before chromatography on aSephadex G-50 column equilibrated in

10 mM Tris-HCI pH 8.0, 0.1 M NaCI and

0.5 mM EDTA. Excluded fractions are mo-

nitored by Cerenkov counting and pooled.

3.3. Colony hybridization

The procedure developped by Grunstein

and Hogness (1975) has made possible toscreen a large number of colonies of E. co/icarrying different hybrid plasmids in order

to determine which plasmids contain a speci-

fied DNA sequence. The colonies to be scree-ned are formed on nitrocellulose filters byreplica plating of a reference set of these co-lonies. After lysing the colonies, their DNAis denatured and fixed to the filter in situ.

The resulting DNA prints of the colonies are

then hybridized to a radioactive RNA or

DNA probe that defines the sequence of inte-

rest, and the result of this hybridization is as-

sayed by autoradiography.

a. Transfer of colonies to nitrocellulose

filters

Following transformation of E. coli com-

petent cells a first screening of recombinantplasmids is carried out by selecting appro-priate antibiotic resistant clones as described

in section 2.10. We will then screen 400 re-

combinant clones (from 4 master LB plates

supplemented with tetracycline) for hybrid

plasmids containing albumin cDNA sequen-

ces. The colonies obtained from the experi-

ment described in Section 2.10 will be trans-

ferred to nitrocellulose filters.

The experimental procedure is a follows:

Step 1. Wash nitrocellulose filters(0.45 ym pores, Schleicher and Schull,

l'ROTOCOLS D'LNGINYERL4 GENETICA 33

BA 85) three times in boiling water for 1 minper wash.

Step 2. Place washed filters between sheetsof 3 MM Whatman paper, autoclave at120°C for 10 min and dry for 10 min in theautoclave.

Step 3. Orientate the filters with a pencilmark and layer on top of the LB master pla-tes. The bacteria are transferred by liftingthe filter once wet from the dish.

Step 4. Place the filters on blotting paper(Whatman 3 MM) saturated with the follow-ing solutions:

- 0.5 M NaOH during 7 min followingby:

- 1 M Tris-HCI pH 7.5 for 2 min follow-ed by:

- 1 M Tris HCI pH 7.5 for 2 min follow-ed by:

- 0.5 M Tris-HCI pH 7.5, 1.5 M NaCl for5 min.

Step 5. Transfer the filters to a sintered-glass funnel and suck dry.

Step 6. Wash filters twice with 100 ml of95 076 ethanol and suck dry.

Step 7. Bake filters under vacuum at 80°C 4.1.for 2 hours.

b. Preparation of nitrocellulose filtersfor hybridization

In order to avoid non-specific binding ofthe labeled probe to the nitrocellulose paper,the filters are prehybridized at 66°C for atleast 6 hours in 3 x SSC (1 x SSC is 0.15 MNaCl, 0.015 M trisodium citrate), 10 xDenhardt's solution (Denhardt's solution is0.002 01o each of Ficoll, polyvinylpyrolidoneand bovine serum albumin), 0.1 01o SDS,150 µg/ml sonicated and denatured E. coliDNA and 500 µg/ml of sonicated and dena-tured salmon sperm DNA. Prehybridation iscarried out in a sealed plastig bag after wet-ting the nitrocellulose filter in 3 x SSC.

c. Hybridization and detection of colonies

containing specific DNA sequences

Following prehybridization, the nitro-cellulose filters are hybridized at 66°C for atleast 24 hours in 10 ml of hybridization solu-tion (3 x SSC, 10 x Denardt's, 0.1 016 SDS,250 yg/ml polyA, 150 pg/ml sonicated anddenatured E. coli DNA). The radioactiveDNA probe to be used is denatured togetherwith E. coli DNA by boiling for 5 min. Afterimmediate cooling in ice-water the DNA pro-be is added to the plastic bag. Use > 106 cpmof labeled DNA per filter.To remove all radioactive probe other

than the specifically hybridized one, filtersare washed extensively as follows:

3 times at 63°C for 30 min each time with100 ml of 2 x SSC, 0.1 07o SDS and 0.1 To Napyrophosphate.

4. Characterization of recombinantplasmids containing albumin cDNAsequences by restriction endonucleasecleavage

Introduction

Plasmid DNA isolated from the positiveclones selected by the in situ colony hybridi-zation screening will be characterized byrestriction enzyme analysis. The size of theinsert will be deduced by comparing thelenght of the recombinant plasmid DNA, li-nearized by EcoRl (which does not cut in therat albumin cDNA sequence and which cutspBR322 only once) to the length of the linearpBR322 DNA on agarose gels. Since the oli-go(dG)-oligo(dC) tailing method used to ge-nerate hybrid plasmids allows reconstitutionof the Pstl site, Pstl restriction cleavage ofthe recombinant plasmid DNA should excisethe inserted sequence. This is an alternativemethod to determine the size of the insert,but several bands might be obtained due tothe presence of several Pstl sites in the albu-

34 JOSEP SIIKL. I S.IL. I i IR LI'.I T

min cDNA sequence. HindIll restrictioncleavage of recombinant plasmids will alsobe performed; this endonuclease cutspBR322 only once but cleaves the rat albu-min cDNA in 3 distant positions. Thisshould allow to orientate the inserted se-quences by reference to the known restric-tion map of rat albumin cDNA.

The detection of specific sequences in

DNA restriction fragments can be done

using the methodology developed by Sout-

hern (J. Mol. Biol. 98:503-517, 1975). The

double-stranded DNA fragments are separa-

ted by size by electrophoresis in an agarose

gel, and the DNA then made single-stranded.

by soaking the gel in alkali. The gel is placed

flat onto a "wick" of filter paper that con-

nects with a trough containing concentrated

salt solution. A single sheet of cellulose

nitrate filter is then placed on top of the gel

and a large stack of dry absorbent paper to-

wels laid flat on top of this. The salt solution

will be drawn up by the absorbent paper to-

wels, passing through the gel and cellulose

nitrate sheet. As the liquid passes through

the gel the single-stranded DNA will be

swept out of the gel and pass onto the

membrane filter. Cellulose nitrate has the

property of binding single-stranded DNA

and so all the DNA will become attached to

this sheet. The final result of this procedure

will be a perfect replica of the DNA from the

original agarose gel, but the DNA single-

stranded and immobilized on a cellulose

nitrate filter sheet. The DNA size pattern,

from the original agarose gel, is faithfully

preserved. Single-stranded DNA bound to a

cellulose nitrate filter is still accessible to hy-

bridization reactions.

We will apply the Southern hybridization

technique to the detection of albumin cDNA

sequences in the EcoRl, Pstl and HindIII

restriction fragments of the selected recom-

binant plasmids. For this purpose the nitro-

cellulose filters will be hybridized to the

32P-labeled albumin cDNA probe or to the

nick- translated pBR322 DNA.

4.2. Rapid preparation of plasmid DNA

from the positive clones

Restriction enzymchanalysis of the recom-binant plasmids does not require highly puri-fied plasmid DNA, such as is obtained bylenghty re-banding in caesium chloride-ethi-

dium bromide gradients. A number of

suitable rapid purification procedures arenow available. The method recently descri-bed by Holmes and Quigley (Analyt.

Biochem. 114:193-197, 1981) can be used.

Inoculate single colonies of the positive re-

combinant clones into sterile test tubes con-taining 5 ml of L broth supplemented with

15 pg/ml tetracycline. Grow overnight at37°C with agitation . Bacteria are collected

by centrifugation in 15 ml Corex tubes and

resuspended in 0.35 ml of 8 °lo sucrose, 5 076triton X-100, 50 mM EDTA, 50 mM Tris-

HCl pH 8.0 and 25 µl of a 10 mg/ml stock of

freshly prepared lysozyme. The solution is

then brought to a boil as rapidly as possible

over a naked flame and maintaned at boiling

point for about 30 to 45 sec in a boiling water

bath. The floculated material is pelleted bycentrifugation at 12,000 g for 10 min at ro-

om temperature. The supernatant is trans-

ferred to an Eppendorf tube and precipitated

by an equal volume of isopropanol at

-20°C for 30 min. The precipitate is pelle-ted by centrifugation in a microfuge at12,000 g for 10 min and resuspended in

100 µl of 10 mM Tris-HC1 pH 8.0, 1 mM

EDTA. Precipitation is repeated twice to ob-

tain clean material for restriction analysis.

4.3. Restriction endonuclease digestions

The pBR322 and recombinant plasmid

DNAs will be digested by the restriction

nucleases EcoRI, HindlIl and PstI.

Restriction endonuclease buffers:

- EcoRl: 100 mM Tris-HCI, pH 7.5;

50 mM NaCl, 5 mM MgCIZ.

- Hindlll: 10 mM Tris-HCI, pH 7.4;

60 mM NaCl; 7 mM MgClz.- Pstl: 6 mM Tris-HCI pH 7.4: 50 mM

NaCl; 6 mM MgCl,; 6 mM 2-mercapto-ethanol.

Mix in a plastic Eppendorf tube:- 10 µl 10 x restriction buffer.

- 5 Ng of plasmid DNA.

- 10 units of restriction enzyme.

- Distilled water up to a final volume of

50 µl.

Incubate at 37°C for 1 hour. Stop thereaction by adding 2µl of 0.5 m EDTA.

4.4 Agarose gel electrophoresis

Restriction fragments are fractionated on

0.8 % or 1.2 % neutral agarose gels. Electro-

phoresis buffer is 10 mM Tris, 5 mM Na ace-

tate, 1 mM EDTA to a final pH 7.9.

Solutions to be prepared:

- 10 x Electrophoresis buffer: 96.91 g

Tris base; 8.2 g Na acetate (anhy-

drous); 7.44 Na, EDTA.

Make up to 2 1 with distilled water. Adjust

to pH 7.9 with glacial acetic acid.

- 10 mg/ml ethidium bromide in H2O.

- Agarose 1.2 °Io in I x electrophoresis

buffer + 5 µg/ 100 ml of ethidium bro-

mide.

- Reservoir buffer.

1 x electrophoresis buffer containing

10µg/ml of ethidium bromide.

Sample preparation: After addition of

EDTA, suplement samples with 10 µl of 10 x

electrophoresis buffer, plus 20 µl of a mixtu-

re containing 20 °lo Ficoll and 0.3 QIo SDS,

plus 20 µl of a 0.1 016 mixture of each bro-

mophenol blue and xylene cyanol FF. Load

20 µl (1 µg of DNA) on gel.Running the gel:

1. Pour in melted agarose.

2. Allow to harden for at least an hour.

Gels can be stored in cold overnight.

3. Run at 50 mA for 3-4 hours. Under

these conditions the bromophenol blue

PROTOCOLS D'L'Nc;1v}LRIA GLSyLTI(1 35

behaves like a 400 bp fragment, appro-

ximately.

4. Observe under U.V. light.

5. Take picture with Polaroid under U.V.

light.

4.5. Transfer of DNA from gels

("Southern blots")

Method of Southern (1975) on nitrocellu-

lose sheets.

Step 1. Prepare the transfer apparatus.

This consists of a tray filled with 10 x SSC

(I x SSC is 0.15 m NaCl, 0.015 M Na citrate

pH 7.0), a glass plate supported on two sides

of the traye, and a thick pad of Whatman

3 MM filter paper soaked in 10 x SSC drap-

per over the glass plate wih two ends dipping

into the solution in the tray.Step 2. Place gel on a tray containing

250 ml of a solution of 1.5 M NaCl and 0.5

N NaOH for 15 min, rocking the tray gently.

Decant solution and add new solution

repeating operation for another 15 min.

Step 3. Carefully decant the alkaline solu-

tion, or draw it off at a water pump, and rin-

se the gel with water to remove residualNaOH.

Step 4. Soak the gel in a neutralizing solu-

tion (250 ml of I M Tris-HCI pH 7.0 con-

taining 1.5 M NaCl) for 20 min. Check pHof solution, if pH > 7.5 repeat treatment on-

ce more for another 20 min.Step. 5. Slide the gel carefully from the

plate on to the pad of filter paper, taking ca-re to avoid trapping air beneith it.

Step 6. Put plastic frame around the gel.This prevents the absorbent paper, which

may sag down, from becoming saturared.

Step 7. Squeeze excess liquid form the sur-face of gel.

Step 8. Take a sheet of nitrocellulose pa-

per (Schleicher and Scholl BA 85) and wet itfirst in water and then in 10 x SSC. Handle

the cellulose nitrate sheet with care, wear

gloves.

36 JO.SEP M,4RIA S,4LA i TREPAT

weight

gel

3 MM wicktransfer buffer

weight

nitrocellulose filter

l /

I

I

^'

/paper towels

'3 MM

gel

/3 MM

Fig. 3. Transfer of DNA from flat gels to cellulose nitrate paper. (a) Stack of paper towels weightedwith a glass plate. (b) Cellulose nitrate paper. (c) Gel surrounded by plastic strips which support the ed-ges of the cellulose paper and the towels. (d) Wad of thick filter paper that dips into the ray of 10 x SSC.(e) Tray of 10 x SSC with a glass plate to support the wad of wet filter paper.

Step 9. Lay the nitrocellulose sheet on thegel, taking care not to trap air beneith it. Theedges of the sheet should be supported by theplastic frame.

Step 10. Soak a piece of Whatman 3 MMpaper in 10 x SSC and lay it on [op of thecellulose nitrate, taking care to avoid trap-ping air beneith it.

Step 11. Stack adsorbent paper on top ofthe 3 MM filter paper and weight it downlightly with a glass plate. Leave the transferovernight (12 h to 24 h).

Step 12. After transfer, carefully removeabsorbent paper and top 3 MM paper. Markthe position and orientation of the gel on thecellulose nitrate sheet with a pencil.

Step 13. Rinse nitrocellulose sheet tho-roughly for 5 to 10 min with 3 x SSC to re-

1

1

move residual agarose particles clinging thegel. This is important as baked agarose willlater lead to important background.

Step 14. Bake nitrocellulose sheet at 80°Cin a vacuum over for 2 hr. The sheet can bestored for many months after baking.

Step 15. Restain gel in ethidium bromide1 µg/ml and observe under U.V. light to en-sure efficient DNA transfer.

4.6. Blot hybridization to rat albumin'1P-cDNA

Before hybridization wash the nitrocellu-lose sheet with 5 x SSC at 65°C for 30 min.Then wash for another 30 min at 65°C with

paper towels

PROTOCOLS D'ENGI.AYLRI,4GLNLTIC/ 37

5 x SSC, 10 x Denhardt's solution (0.2 %

w/v of each Ficoll M.W. 400,000, polyvinyl-

pyrollidone M.W. 360,000 and bovine serum

albumin).

After washing prehybridize the sheet at

67°C for a minimum of 4 hr in a solution

containing: 5 x SSC; 10 x Denhardt's solu-

tion; 50 mM Tris-HC1, pH 7.5; 1 mM

EDTA; 0.1 01o SDS and 100 µg/ml of sonica-

ted and denatured salmon sperm DNA. Fi-

nally hybridize at 67°C for 24 to 48 h in

10 ml of the same solution to which the hy-

bridization probe is added after denatu-

ration.

After hybridization, the nitrocellulose

sheet is washed step-wise as follow:

- 30 min at 67°C with 250 ml of 5 x SSC;

10 x Denhardt's solution; 0.1 % SDS

and 0.1 01o Na pyrophosphate.

- 30 min at 65°C with 250 ml of 3 x SSC;

10 x Denhardt's solution; 0.1 01o SDS

and 0.1 01o Na pyrophosphate.

-4x30minat63°C with 250 ml of 2 x

SSC; 1 x Denhardt's solution; 0.1 010

SDS and 0.1 016 Na pyrophosphate.

- 2 x 30 min at 63°C with 250 ml of 1 x

SSC; 1 x Denhardt's solution; 0.1 076SDS and 0.1 01o Na pyrophospate.

- 20 min at 63°C with 250 ml of 0.7 x

SSC; 0.1 % SDS and 0.1 To Na pyro-

phosphate.

After washing the filter it is blotted dry

with Whatman 3 MM paper and exposed to

an X-ray film (Kodak X-omat) with inten-

sifying screens (Dupon Cronex Lightning

Plus) at -70°C.

5. Addendum

5.1. Buffers

Column buffer cDNA synthesis:

- 20 mM Na acetate pH 5.5.

- 0.2 M LiCI.

- 1 mM EDTA.

Column buffer Nick-translation:

- 10 mM Tris-HCI pH 8.0.

- 0.1 M NaCl.

- 0.5 mM EDTA.

Tailing buffer: 10 x

- 1.4 M Na cacodylate.

- 0.3 M Tris.

- 1 mM DTT.

The pH should be 7. 6, but will drop down

to 6.9 upon dilution.

Annealing buffer:

- 10 mM Tris-HCI pH 8.1.

-0.1 MNaCI.

- 0.2 mM EDTA.

Restriction endonucleases buffers:

EcoRl : 100 mM Tris - HCI pH 7.5; 50 mM

NaCl; 5 mM MgCl2.

Hindlll : 10 mM Tris-HCI , pH 7.4, 60 mM

NaCl; 7 mM MgC12.

Pstl: 6 mM Tris -HCI, pH 7.4; 50 mM

NaCl; 6 mM MgCIZ 6 mM 2- mercapto-

ethanol.Prepare 10 x concentrated for digestions.

5.2. Culture media

L Broth contains NaCl (10 g), Difco Yeast-

Extract (5 g), Difco Bacto Tryptone (10 g)

and distilled water in a total volume of 1 I.

Dissolve. Adjust pH to 7.0 with 2 M NaOH.

Autoclave at 120°C for 20 min.

LB medium for plates contains I I of

L broth and 15 g of Difco Bacto Agar added

before autoclaving.Antibiotic containing media: L broth of

LB medium for plates is cooled to 60°C after

autoclaving and supplemented with tetracy-

cline (15 Ng/ml) or ampicillin (10 pg/ml)

from stock solutions (2 mg/ml for tetra-

cycline and 20 mg/ml for ampicillin) sterili-

zed by Millipore filtration.

Z broth contains 1.6 g Nutrient broth, 1 g

38 JOSEP MARL! SALA i TREPAT

5.3 Restriction map of pBR322 DNA

(2.6 x 106 d.)

Nde 12297 / 1 ( \ '., 2031r1;) E i 2295

A__^ • 2246 I V `Pvu II 2066

Sna 1 '246 Tth 111 I 2219

Ball 1444

0 2 4 6 8 10 12 14 16 13 20

b ^ E3 F3 a a CaW 1"I NH U~ W U

NC C

C N NC C

rnC

M 2 Q S Cr CL a_ S I Cr m iI I I 1 I I I 1 1 I I 1

RSA 510 RSA 1311

4- a

RSA 57

o- C o-^---a O-

H o-O

BamH I 375

Ban II 471

0

•--^ a-O----u 1--+ ^-s

PROTO('OLS D'ENGIN1ERl^1 GENCTl('4 39

peptone, 0.2 g glucose and 100 ml H20. Dis-solve. Adjust to pH 7.5 with 2 M NaOH be-fore autoclaving.

5.4. Safety considerations:

physical containment

Although the genetic engineering experi-

ments that will be performed are of null po-

tential risk, special attention has to be paid

in order to avoid that the E. coli recombi-

nant clones obtained escape to the external

environment. Work with this material will becarried out in a limited area and all cultureswill be immediately autoclaved after use.

6. BIBLIOGRAPHY

Books of general interest.Genetic Engineering (1981). Wu.ttAMSON, R. ed.; Vols.

I and 11. Academic Press London - New York.Principles of Gene Manipulation. An Introduction to

Genetic Engineering (1980), OLD, R.W. and PRiMRO-sE, S.B. Blackwell Scientific Publications, Oxford.

Genetic Engineering - Cloning DNA (1980), GLOVER,D.M. Chapman and Hall. London - New York.

Genetic Engineering (1978), CHAKRABARTY, A.M., ed.,

CRC Press, Inc. West Palm Beach, Florida.Genetic Engineering. Principles and Methods (1979/

1980), SETLOW, J.K. and Hot IAENDER, A. eds.; vols.

I and ll. Plenum Press. New York and London.

Methods in Enzymology. Recombinant DNA (1979),Vol. 68, Wu, R. ed.; Academic Press London-NewYork.

Methods in Enzymology. Nucleic Acids. Part 1 (1980),vol. 65, GROSSMAN, L. and MOLDAVE, K. eds.; Acade-

mic Press. London-New York.